Close interaction between cells (compaction) is needed for the formation of the junctions that bind cells together to form an epithelium. The process of epithelium formation can be studied in a model system using F9 embryonal carcinoma (EC) cells. In aggregate cultures of the undifferentiated stem cells, the cells compact tightly, and after four days of induction by retinoic acid in the medium, an outer epithelial layer is observed. A mutant F9 cell line (F9att-5.51), does not compact in suspension cultures and will not differentiate into epithelial structures. The mutant line will not grow as a nomolayer because it does not attch to substrates. Although the membrane glycoprotein uvomorulin (UM, E-cadherin) is reduced in the mutant, the introduction of excess UM into the mutant cells did not rescue the mutant behavior and therefore cannot account for the defects in this line. We have now identified a component in the compaction mechanism that is likely to be the primary cause of all the adhesive defects in the mutant cell line. The cytoskeletal protein vinculin is completely absent from mutant cells. Vinculin is known to be involved both in cell-cell interactions and specifically in zonula adherens junctions in epithelial cells as well as in cell-substrate adhesion plaques. The absence of this protein could therefore account for the adhesive defects in 5.51 cells. This will be tested by the transfection of an expression vector for vinculin and for deleted versions that can be used to determine the roles of the domains of vinculin in adhesion. We will identify and characterize the 5' regulatory sequences of the gene. The second major aim is to study the other components in multistep adhesion processes by cell fractionation and affinity chromatography using the contrasting F9 cell lines that already exist and those that will be constructed during the research perioc. This proposal exploits the exostence of the mutant cell line to explore the structure-function domains of vinculin and the interactions between adhesive components in the processes that ensure epithelial function and stability. Knowledge of adhesive components is important in addressing problems of metastases in carcinoma as well as understanding the regulation of the cytoskeleton and how it relates to cell behavior.